Thermostable Alpha-Amylases

ABSTRACT

The present invention relates to an isolated polynucleotide comprising an open reading frame encoding a polypeptide having alpha-amylase activity, the polypeptide selected from the group consisting of:
         a) a polypeptide comprising an amino acid sequence which has at least 70% identity with amino acids 22 to 450 of SEQ ID NO: 4;   b) a polypeptide comprising an amino acid sequence which has at least 70% identity with the polypeptide encoded by the amylase encoding part of the polynucleotide inserted into a plasmid present in the  E. coli  host deposited under the Budapest Treaty with DSMZ under accession number DSM 15334;   c) a polypeptide encoded by a polynucleotide comprising a nucleotide sequence which has at least 70% identity with the sequence shown from position 68 to 1417 in SEQ ID NO: 3; and   d) a fragment of (a), (b) or (c) that has alpha-amylase activity.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.13/230,984 filed Sep. 13, 2011, which is a divisional of U.S.application Ser. No. 12/755,076 filed on Apr. 6, 2010, now U.S. Pat. No.8,039,241, which is a continuation of U.S. application Ser. No.12/036,806 filed Feb. 25, 2008, now abandoned, which is a divisional ofU.S. application Ser. No. 11/671,692 filed Feb. 6, 2007, now abandoned,which is a divisional of U.S. application Ser. No. 10/539,396 filed Jun.16, 2005, now U.S. Pat. No. 7,189,552, which is a national phaseapplication under 35 U.S.C. 371 of PCT/DK2003/00882 filed Dec. 16, 2003,which claims priority or the benefit under 35 U.S.C. 119 of Danishapplication no. PA 2002 01928 filed Dec. 17, 2002 and U.S. provisionalapplication No. 60/435,483 filed Dec. 20, 2002, the contents of whichare fully incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to thermostable alpha-amylases, inparticular with improved thermal stability at acidic pH. The inventionalso relates to the use of such alpha-amylases.

BACKGROUND OF THE INVENTION

Alpha-Amylases (alpha-1,4-glucan-4-glucanohydrolases, EC. 3.2.1.1)constitute a group of enzymes which catalyze hydrolysis of starch andother linear and branched 1,4-glucosidic oligo- and polysaccharides.

There is a very extensive body of patent and scientific literaturerelating to this industrially very important class of enzymes. A numberof alpha-amylases referred to as “Termamyl®-like alpha-amylases” andvariants thereof are known from, e.g., WO 90/11352, WO 95/10603, WO95/26397, WO 96/23873 and WO 96/23874. Termamyl®-like alpha-amylases arevery thermostable and therefore suitable for processes carried out athigh temperatures such as starch liquefaction in dextrose productionprocesses.

Another group of alpha-amylases are referred to as “Fungamyl™-likealpha-amylases”, which are alpha-amylases related or homologous to thealpha-amylase derived from Aspergillus oryzae. The Fungamyl™-likealpha-amylases have a relatively low thermostability the commercialproduct sold under the tradename FUNGAMYL™ by Novozymes A/S, Denmark,has a optimum around 55° C., and is not suitable for processes carriedout at high temperatures. Fungamyl™-like alpha-amylases are today usedfor making syrups for, e.g., the brewing industry.

Clearly, it would be advantageous to provide an alpha-amylase withincreased thermostability preferably at an acidic pH. This is no newrealization, but actually a very long-felt need in the art. As far backas in 1980, Somkuti and Steinberg described a thermoacidophilicextracellular alpha-amylase of Rhizomucor pusillus (Mucor pusillus),that they managed to isolate and characterize. They state that: “Sincehigh temp and acidic pH are optimum conditions for the economichydrolysis of starch, the use of thermostable and acid-stable amylasesof microbial origin for industrial purposes has been recommended”, andgo on to conclude about the Rhizomucor amylase that: “It is apparentlythe first example of fungal alpha-amylase exhibiting both acidophily andthermophily simultaneously. Consequently, the alpha-amylase of M.pusillus should be of economic importance.” (Somkuti and Steinberg,1980, “Thermoacidophilic extracellular amylase of Mucor pusillus”, Dev.Indust. Microbiol. 21:327-337).

However, despite the very clear conclusions by Somkuti and Steinbergback in 1980, the gene encoding the Rhizomucor pusillus alpha-amylasehad until today not been cloned or sequenced, and the amylase had untiltoday not been produced recombinantly in industrially relevant amounts.In 1987 an improved purification method was reported, but still only forenzyme produced by the wild-type Rhizomucor pusillus (Turchi and Becker,1987, Curr. Microbiol. 15:203-205).

SUMMARY OF THE INVENTION

A problem to be solved by this invention is how to provide a recombinantthermoacidophilic alpha-amylase. The present inventors have successfullyisolated a gene from Rhizomucor pusillus encoding an alpha-amylase whichthey have denoted AM782, they have successfully introduced the encodinggene into a recombinant industrial filamentous fungal expression system,and produced the alpha-amylase. Characterization of the amylase hasshown it to be a highly thermoacidophilic alpha-amylase which has ahighly interesting activity as demonstrated by the sugar profile frommaltodextrin hydrolysis by amylase AM782.

The amylase AM782 can work at a very high temperature, at least up to70° C. The amylase AM782 has a very fast reaction speed; when comparedat the same dosage with Fungamyl™ 800 L, the amylase AM782 can achievein about 3 hours, what takes Fungamyl™ 24 to 48 hours. Furthermore, theamylase AM782 can degrade DP3 into DP2 and DP1, so it gives a higher DP1result.

Accordingly, in a first aspect the invention relates to an isolatedpolynucleotide comprising an open reading frame encoding a polypeptidehaving alpha-amylase activity, the polypeptide selected from the groupconsisting of: a) a polypeptide comprising an amino acid sequence whichhas at least 70% identity with amino acids 22 to 450 of SEQ ID NO: 4,preferably 75%, more preferably 80%, even more preferably 85%, stillmore preferably 90%, more preferably 95%, and most preferably at least97% identity with amino acids 22 to 450 of SEQ ID NO: 4; b) apolypeptide comprising an amino acid sequence which has at least 70%identity with the polypeptide encoded by the amylase encoding part ofthe polynucleotide inserted into a plasmid present in the E. coli hostdeposited under the Budapest Treaty with DSMZ under accession number DSM15334, preferably 75%, more preferably 80%, even more preferably 85%,still more preferably 90%, more preferably 95%, and most preferably atleast 97% identity with the polypeptide encoded by the amylase encodingpart of the polynucleotide inserted into a plasmid present in the E.coli host deposited under the Budapest Treaty with DSMZ under accessionnumber DSM 15334; c) a polypeptide encoded by a polynucleotidecomprising a nucleotide sequence which has at least 70% identity withthe sequence shown from position 68 to 1417 in SEQ ID NO: 3, preferably75%, more preferably 80%, even more preferably 85%, still morepreferably 90%, more preferably 95%, and most preferably at least 97%identity with the sequence shown from position 68 to 1417 in SEQ ID NO:3; and d) a fragment of (a), (b) or (c) that has alpha-amylase activity.

In a second aspect the invention relates to a nucleic acid constructcomprising a polynucleotide as defined in the first aspect operablylinked to one or more control sequences that direct the production ofthe polypeptide in a suitable host.

A third aspect relates to a recombinant expression vector comprising anucleic acid construct as defined in the second aspect.

In a fourth aspect the invention relates to a recombinant host cellcomprising a nucleic acid construct as defined the second aspect, or atleast one copy of an expression vector as defined in the third aspect.

Industrial production of the amylase AM782 along with homologues andvariants is of course highly interesting.

Accordingly, in a fifth aspect the invention relates to a method forproducing a polypeptide having alpha-amylase activity encoded by apolynucleotide as defined in the first aspect, the method comprising:(a) cultivating a recombinant host cell as defined in any of claims12-16 under conditions conducive for production of the polypeptide; and(b) recovering the polypeptide.

There are quite a few applications for an amylase such as the amylaseAM782, an overview of some of the major ones is given herein, and itincludes but is not limited to: the starch industry, the food processingindustry, the textile industry, and the detergent industry.

Consequently, additional aspects of the invention relate to a method ofproducing an enzymatically modified starch derivative, wherein apolypeptide having alpha-amylase activity produced according to a methodas defined in the fifth aspect is used for liquefying and/orsaccharifying starch; and to a method of producing high maltose syrups,wherein a polypeptide having alpha-amylase activity produced accordingto a method as defined in the fifth aspect is used for liquefyingstarch; a method for desizing textile, wherein a polypeptide havingalpha-amylase activity produced according to a method as defined in thefifth aspect is used for treating the textile; and to a brewing process,wherein a polypeptide having alpha-amylase activity produced accordingto a method as defined in the fifth aspect is added during fermentationof wort; and to an alcohol production process, wherein a polypeptidehaving alpha-amylase activity produced according to a method as definedin the fifth aspect is used for liquefaction starch in a distillerymash; and to a process, wherein a dough product comprising a polypeptidehaving alpha-amylase activity produced according to a method as definedin the fifth aspect is baked.

Various uses of amylase AM782 along with homologues and variants arealso contemplated in the present invention.

Accordingly, a number of non-limiting aspects of the invention relate tothe use of a polypeptide having alpha-amylase activity producedaccording to a method as defined in the fifth aspect in a starchconversion process for liquefaction and/or saccharification; to the useof a polypeptide having alpha-amylase activity produced according to amethod as defined in the fifth aspect for liquefying starch in a highmaltose syrup production process; to the use of a polypeptide havingalpha-amylase activity produced according to a method as defined in thefifth aspect for textile desizing; to the use of a polypeptide havingalpha-amylase activity produced according to a method as defined in thefifth aspect for producing alcohol; to the use of a polypeptide havingalpha-amylase activity produced according to a method as defined in thefifth aspect for brewing; and to the use of a polypeptide havingalpha-amylase activity produced according to a method as defined in thefifth aspect for baking.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the pH profiles of the amylases AM782, FUNGAMYL™ and BAN.

FIG. 2 shows the temperature profiles of the amylases AM782, FUNGAMYL™and BAN.

FIG. 3-1 shows the stability of the amylases AM782, FUNGAMYL™ and BAN atpH 5.0 at 60° C.

FIG. 3-2 shows the stability of the amylases AM782, FUNGAMYL™ and BAN atpH 5.0 at 70° C.

FIG. 3-3 shows the stability of the amylases AM782, FUNGAMYL™ and BAN atpH 5.0 at 80° C.

FIG. 4-1 shows the stability of the amylases AM782, FUNGAMYL™ and BAN atpH 5.0 at 60° C.

FIG. 4-2 shows the stability of the amylases AM782, FUNGAMYL™ and BAN atpH 5.0 at 70° C.

FIG. 4-3 shows the stability of the amylases AM782, FUNGAMYL™ and BAN atpH 5.0 at 80° C.

FIG. 5-1 shows the stability of the amylases AM782, FUNGAMYL™ and BAN atpH 5.0 at 60° C.

FIG. 5-2 shows the stability of the amylases AM782, FUNGAMYL™ and BAN atpH 5.0 at 70° C.

FIG. 5-3 shows the stability of the amylases AM782, FUNGAMYL™ and BAN atpH 5.0 at 80° C.

FIG. 6 shows the Aspergillus expression vector pDAu71.

FIG. 7 shows the amylase expression vector pPFJo143.

FIG. 8 shows results of the sugar profile of maltodextrin hydrolysis bythe alpha-amylase AM782.

DEFINITIONS Sequence Homology and Alignment

For purposes of the present invention, alignments of sequences andcalculation of homology scores may be done using a full Smith-Watermanalignment, useful for both protein and DNA alignments. The defaultscoring matrices BLOSUM50 and the identity matrix are used for proteinand DNA alignments respectively. The penalty for the first residue in agap is −12 for proteins and −16 for DNA, while the penalty foradditional residues in a gap is −2 for proteins and −4 for DNA.Alignment may be made with the FASTA package version v20u6 (Pearson andLipman, 1988, “Improved Tools for Biological Sequence Analysis”, PNAS85:2444-2448, and Pearson, 1990, “Rapid and Sensitive SequenceComparison with FASTP and FASTA”, Methods in Enzymology 183:63-98).

Multiple alignments of protein sequences may be made using “ClustalW”(Thompson, Higgins, and Gibson, 1994, “CLUSTAL W: improving thesensitivity of progressive multiple sequence alignment through sequenceweighting, positions-specific gap penalties and weight matrix choice”,Nucleic Acids Research 22:4673-4680). Multiple alignment of DNAsequences may be done using the protein alignment as a template,replacing the amino acids with the corresponding codon from the DNAsequence.

Substantially Pure Polynucleotide

The term “substantially pure polynucleotide” as used herein refers to apolynucleotide preparation, wherein the polynucleotide has been removedfrom its natural genetic milieu, and is thus free of other extraneous orunwanted coding sequences and is in a form suitable for use withingenetically engineered protein production systems. Thus, a substantiallypure polynucleotide contains at the most 10% by weight of otherpolynucleotide material with which it is natively associated (lowerpercentages of other polynucleotide material are preferred, e.g., at themost 8% by weight, at the most 6% by weight, at the most 5% by weight,at the most 4% at the most 3% by weight, at the most 2% by weight, atthe most 1% by weight, and at the most ½% by weight). A substantiallypure polynucleotide may, however, include naturally occurring 5′ and 3′untranslated regions, such as promoters and terminators. It is preferredthat the substantially pure polynucleotide is at least 92% pure, i.e.,that the polynucleotide constitutes at least 92% by weight of the totalpolynucleotide material present in the preparation, and higherpercentages are preferred such as at least 94% pure, at least 95% pure,at least 96% pure, at least 96% pure, at least 97% pure, at least 98%pure, at least 99%, and at the most 99.5% pure. The polynucleotidesdisclosed herein are preferably in a substantially pure form. Inparticular, it is preferred that the polynucleotides disclosed hereinare in “essentially pure form”, i.e., that the polynucleotidepreparation is essentially free of other polynucleotide material withwhich it is natively associated. Herein, the term “substantially purepolynucleotide” is synonymous with the terms “isolated polynucleotide”and “polynucleotide in isolated form”.

cDNA

The term “cDNA” when used in the present context, is intended to cover aDNA molecule which can be prepared by reverse transcription from amature, spliced, mRNA molecule derived from a eukaryotic cell. cDNAlacks the intron sequences that are usually present in the correspondinggenomic DNA. The initial, primary RNA transcript is a precursor to mRNAand it goes through a series of processing events before appearing asmature spliced mRNA. These events include the removal of intronsequences by a process called splicing. When cDNA is derived from mRNAit therefore lacks intron sequences.

DETAILED DESCRIPTION OF THE INVENTION

The first aspect of the invention relates to an isolated polynucleotidecomprising an open reading frame encoding a polypeptide havingalpha-amylase activity, the polypeptide selected from the groupconsisting of: a) a polypeptide comprising an amino acid sequence whichhas at least 70% identity with amino acids 22 to 450 of SEQ ID NO: 4; b)a polypeptide comprising an amino acid sequence which has at least 70%identity with the polypeptide encoded by the amylase encoding part ofthe polynucleotide inserted into a plasmid present in the E. coli hostdeposited under the Budapest Treaty with DSMZ under accession number DSM15334; c) a polypeptide encoded by a polynucleotide comprising anucleotide sequence which has at least 70% identity with the sequenceshown from position 68 to 1417 in SEQ ID NO: 3; and d) a fragment of(a), (b) or (c) that has alpha-amylase activity.

The techniques used to isolate or clone a nucleotide sequence encoding apolypeptide are known in the art and include isolation from genomic DNA,preparation from cDNA, or a combination thereof. The cloning of thenucleotide sequences of the present invention from such genomic DNA canbe effected, e.g., by using the well known polymerase chain reaction(PCR) or antibody screening of expression libraries to detect cloned DNAfragments with shared structural features. See, e.g., Innis et al.,1990, PCR: A Guide to Methods and Application, Academic Press, New York.Other amplification procedures such as ligase chain reaction (LCR),ligated activated transcription (LAT) and nucleotide sequence-basedamplification (NASBA) may be used. The nucleotide sequence may be clonedfrom a strain of Rhizomucor, or another related organism and thus, forexample, may be an allelic or species variant of the polypeptideencoding region of the nucleotide sequence.

The nucleotide sequence may be obtained by standard cloning proceduresused in genetic engineering to relocate the nucleotide sequence from itsnatural location to a different site where it will be reproduced. Thecloning procedures may involve excision and isolation of a desiredfragment comprising the nucleotide sequence encoding the polypeptide,insertion of the fragment into a vector molecule, and incorporation ofthe recombinant vector into a host cell where multiple copies or clonesof the nucleotide sequence will be replicated. The nucleotide sequencemay be of genomic, cDNA, RNA, semisynthetic, synthetic origin, or anycombinations thereof.

The term “polypeptide variant”, “protein variant”, “enzyme variant”, orsimply “variant” refers to a polypeptide of the invention comprising oneor more alteration(s), such as substitution(s), insertion(s),deletion(s), and/or truncation(s) of one or more specific amino acidresidue(s) in one or more specific position(s) in the polypeptide. Thetotal number of such alterations is typically not more than 10, e.g.,one, two, three, four, five, six, seven, eight, or nine of saidalterations. In addition, the variant of the invention may include othermodifications of the parent enzyme, typically not more than 10, e.g.,not more than 5 such modifications. The variant generally has a degreeof sequence identity with the parent polypeptide of at least 80%, e.g.,at least 85%, typically at least 90%, or at least 95%.

The term “parent polypeptide”, “parent protein”, “parent enzyme”,“standard enzyme”, or simply “parent” refers to the polypeptide on whichthe variant was based. This term also refers to the polypeptide withwhich a variant is compared and aligned. The parent may be a naturallyoccurring (wild-type) polypeptide, or it may in turn even be a variantthereof, prepared by any suitable means. For instance, the parentprotein may be a variant of a naturally occurring polypeptide which hasbeen modified or altered in the amino acid sequence. A parent may alsobe an allelic variant which is any of two or more alternative forms of agene occupying the same chromosomal locus. Allelic variation arisesnaturally through mutation, and may result in polymorphism withinpopulations as is well-described in the art. An allelic variant of apolypeptide is a polypeptide encoded by the corresponding allelicvariant of a gene.

The term “randomized library”, “variant library”, or simply “library”refers to a library of variant polypeptides. Diversity in the variantlibrary can be generated via mutagenesis of the genes encoding thevariants at the DNA triplet level, such that individual codons arevariegated, e.g., by using primers of partially randomized sequence in aPCR reaction. Several techniques have been described, by which one cancreate a diverse combinatorial library by variegating several nucleotidepositions in a gene and recombining them, for instance where thesepositions are too far apart to be covered by a single (spiked or doped)oligonucleotide primer. These techniques include the use of in vivorecombination of the individually diversified gene segments as describedin WO 97/07205 on page 3, lines 8 to 29 (Novozymes A/S). They alsoinclude the use of DNA shuffling techniques to create a library of fulllength genes, wherein several gene segments are combined, and whereineach segment may be diversified, e.g., by spiked mutagenesis (Stemmer,1994, Nature 370: 389-391, and U.S. Pat. Nos. 5,605,793; 5,811,238; and5,830,721). One can use a gene encoding a protein “backbone” (wild-typeparent polypeptide) as a template polynucleotide, and combine this withone or more single or double-stranded oligonucleotides as described inWO 98/41623 and in WO 98/41622 (Novozymes A/S). The single-strandedoligonucleotides could be partially randomized during synthesis. Thedouble-stranded oligonucleotides could be PCR products incorporatingdiversity in a specific region. In both cases, one can dilute thediversity with corresponding segments encoding the sequence of thebackbone protein in order to limit the average number of changes thatare introduced.

Methods have also been established for designing the ratios ofnucleotide mixtures (A; C; T; G) to be inserted in specific codonpositions during oligo- or polynucleotide synthesis, so as to introducea bias in order to approximate a desired frequency distribution towardsa set of one or more desired amino acids that will be encoded by theparticular codons. It may be of interest to produce a variant librarythat comprises permutations of a number of known amino acidmodifications in different locations in the primary sequence of thepolypeptide. These could be introduced post-translationally or bychemical modification sites, or they could be introduced throughmutations in the encoding genes. The modifications by themselves maypreviously have been proven beneficial for one reason or another (e.g.,decreasing antigenicity, or improving specific activity, performance,stability, or other characteristics). In such instances, it may bedesirable first to create a library of diverse combinations of knownsequences. For example, if twelve individual mutations are known, onecould combine (at least) twelve segments of the parent protein encodinggene, wherein each segment is present in two forms: one with, and onewithout the desired mutation. By varying the relative amounts of thosesegments, one could design a library (of size 212) for which the averagenumber of mutations per gene can be predicted. This can be a useful wayof combining mutations, that by themselves give some, but not sufficienteffect, without resorting to very large libraries, as is often the casewhen using ‘spiked mutagenesis’. Another way to combine these ‘knownmutations’ could be by using family shuffling of oligomeric DNA encodingthe known mutations with fragments of the full length wild typesequence.

Accordingly, a preferred embodiment of the invention relates to apolynucleotide of the first aspect, wherein the polypeptide is anartificial variant comprising an amino acid sequence that has one ormore truncation(s), and/or at least one substitution, deletion, and/orinsertion of an amino acid as compared to amino acids 22 to 450 of SEQID NO: 4.

It will be apparent to those skilled in the art that such modificationscan be made outside the regions critical to the function of the moleculeand still result in an active polypeptide. Amino acid residues essentialto the activity of the polypeptide encoded by the nucleotide sequence ofthe invention, and therefore preferably not subject to modification,such as substitution, may be identified according to procedures known inthe art, such as site-directed mutagenesis or alanine-scanningmutagenesis (see, e.g., Cunningham and Wells, 1989, Science 244:1081-1085). In the latter technique, mutations are introduced at everypositively charged residue in the molecule, and the resultant mutantmolecules are tested for amylase activity to identify amino acidresidues that are critical to the activity of the molecule. Sites ofsubstrate-enzyme interaction can also be determined by analysis of thethree-dimensional structure as determined by such techniques as nuclearmagnetic resonance analysis, crystallography or photoaffinity labelling(see, e.g., de Vos et al., 1992, Science 255: 306-312; Smith et al.,1992, Journal of Molecular Biology 224: 899-904; Wlodaver et al., 1992,FEBS Letters 309: 59-64).

Moreover, a nucleotide sequence encoding a polypeptide of the presentinvention may be modified by introduction of nucleotide substitutionswhich do not give rise to another amino acid sequence of the polypeptideencoded by the nucleotide sequence, but which correspond to the codonusage of the host organism intended for production of the enzyme.

The introduction of a mutation into the nucleotide sequence to exchangeone nucleotide for another nucleotide may be accomplished bysite-directed mutagenesis using any of the methods known in the art.Particularly useful is the procedure, which utilizes a supercoiled,double stranded DNA vector with an insert of interest and two syntheticprimers containing the desired mutation. The oligonucleotide primers,each complementary to opposite strands of the vector, extend duringtemperature cycling by means of Pfu DNA polymerase. On incorporation ofthe primers, a mutated plasmid containing staggered nicks is generated.Following temperature cycling, the product is treated with DpnI which isspecific for methylated and hemimethylated DNA to digest the parentalDNA template and to select for mutation-containing synthesized DNA.Other procedures known in the art may also be used. For a generaldescription of nucleotide substitution, see, e.g., Ford et al., 1991,Protein Expression and Purification 2: 95-107.

Another preferred embodiment relates to a polynucleotide of the firstaspect, wherein the polypeptide comprises an amino acid sequence whichhas at least 70% identity with amino acids 22 to 450 of SEQ ID NO: 4,preferably at least 75%, more preferably 80%, still more preferably 85%,still even more preferably 90%, more preferably 95%, and most preferablyat least 97% identity with amino acids 22 to 450 of SEQ ID NO: 4.

Yet another preferred embodiment relates to a polynucleotide of thefirst aspect, wherein the polypeptide comprises the amino acids 22 to450 of SEQ ID NO: 4.

In a preferred embodiment the invention relates to a polynucleotide ofthe first aspect, wherein the polypeptide consists of the amino acids 22to 450 of SEQ ID NO: 4.

Still another preferred embodiment the invention relates to apolynucleotide of the first aspect, wherein the polypeptide comprises anamino acid sequence which has at least 70% identity with the polypeptideencoded by the amylase encoding part of the nucleotide sequence insertedinto a plasmid present in the E. coli host deposited under the BudapestTreaty with DSMZ under accession number DSM 15334, preferably at least80% identity, at least 85% identity, at least 90% identity, at least 95%identity, or at least 97% identity with the polypeptide encoded by theamylase encoding part of the nucleotide sequence inserted into a plasmidpresent in the E. coli host deposited under the Budapest Treaty withDSMZ under accession number DSM 15334; preferably the polypeptidecomprises the amino acid sequence encoded by the amylase encoding partof the nucleotide sequence inserted into a plasmid present in the E.coli host deposited under the Budapest Treaty with DSMZ under accessionnumber DSM 15334; still more preferably the polypeptide consists of theamino acid sequence encoded by the amylase encoding part of thenucleotide sequence inserted into a plasmid present in the E. coli hostdeposited under the Budapest Treaty with DSMZ under accession number DSM15334.

Another preferred embodiment relates to the polynucleotide of the firstaspect, wherein the polypeptide is an artificial variant which comprisesan amino acid sequence that has one or more truncation(s), and/or atleast one substitution, deletion, and/or insertion of an amino acid ascompared to the amino acid sequence encoded by the amylase encoding partof the nucleotide sequence inserted into a plasmid present in the E.coli host deposited under the Budapest Treaty with DSMZ under accessionnumber DSM 15334.

Nucleic Acid Construct

When used herein, the term “nucleic acid construct” means a nucleic acidmolecule, either single- or double-stranded, which is isolated from anaturally occurring gene or which has been modified to contain segmentsof nucleic acids in a manner that would not otherwise exist in nature.

The term nucleic acid construct is synonymous with the term “expressioncassette” when the nucleic acid construct contains the control sequencesrequired for expression of a coding sequence of the present invention. Apolynucleotide sequence encoding a polypeptide of the present inventionmay be manipulated in a variety of ways to provide for expression of thepolypeptide. Manipulation of the nucleotide sequence prior to itsinsertion into a vector may be desirable or necessary depending on theexpression vector. The techniques for modifying nucleotide sequencesutilizing recombinant DNA methods are well known in the art.

The term “control sequences” is defined herein to include allcomponents, which are necessary or advantageous for the expression of apolypeptide of the present invention. Each control sequence may benative or foreign to the nucleotide sequence encoding the polypeptide.Such control sequences include, but are not limited to, a leader,polyadenylation sequence, propeptide sequence, promoter, signal peptidesequence, and transcription terminator. At a minimum, the controlsequences include a promoter, and transcriptional and translational stopsignals. The control sequences may be provided with linkers for thepurpose of introducing specific restriction sites facilitating ligationof the control sequences with the coding region of the nucleotidesequence encoding a polypeptide.

The term “operably linked” is defined herein as a configuration in whicha control sequence is appropriately placed at a position relative to thecoding sequence of the DNA sequence such that the control sequencedirects the expression of a polypeptide.

When used herein the term “coding sequence” is intended to cover anucleotide sequence, which directly specifies the amino acid sequence ofits protein product. The boundaries of the coding sequence are generallydetermined by an open reading frame, which usually begins with the ATGstart codon. The coding sequence typically includes DNA, cDNA, andrecombinant nucleotide sequences.

An aspect of the invention relates to a nucleic acid constructcomprising a polynucleotide as defined in the first aspect operablylinked to one or more control sequences that direct the production ofthe polypeptide in a suitable host.

Expression Vector

In the present context, the term “expression” includes any step involvedin the production of the polypeptide including, but not limited to,transcription, post-transcriptional modification, translation,post-translational modification, and secretion.

In the present context, the term “expression vector” covers a DNAmolecule, linear or circular, that comprises a segment encoding apolypeptide of the invention, and which is operably linked to additionalsegments that provide for its transcription.

An aspect of the invention relates to a recombinant expression vectorcomprising a nucleic acid construct as defined in the previous aspect.

According to the invention, a polynucleotide encoding an amylase of theinvention can be expressed using an expression vector which typicallyincludes control sequences such as a promoter, an operator, a ribosomebinding site, a translation initiation signal, and optionally arepressor gene, or various activator genes. The recombinant expressionvector carrying the polynucleotide encoding an alpha-amylase of theinvention may be any vector which can be subjected to recombinant DNAprocedures, and the choice of vector will often depend on the host cellinto which it is to be introduced. The vector may be one which, whenintroduced into a host cell, is integrated into the host cell genome andreplicated together with the chromosome(s) into which it has beenintegrated. Examples of suitable expression vectors include pMT838.

In the vector, the DNA sequence should be operably connected to asuitable promoter sequence. The promoter may be any DNA sequence, whichshows transcriptional activity in the host cell of choice and may bederived from genes encoding proteins either homologous or heterologousto the host cell.

The expression vector of the invention may also comprise a suitabletranscription terminator and, in eukaryotes, polyadenylation sequencesoperably connected to the DNA sequence encoding the alpha-amylasevariant of the invention. Termination and polyadenylation sequences maysuitably be derived from the same sources as the promoter.

The vector may further comprise a DNA sequence enabling the vector toreplicate in the host cell in question. Examples of such sequences arethe origins of replication of plasmids pUC19, pACYC177, pUB110, pE194,pAMB1 and pIJ702.

The vector may also comprise a selectable marker, e.g., a gene theproduct of which complements a defect in the host cell, such as the dalgenes from B. subtilis or B. licheniformis, or one which confersantibiotic resistance such as ampicillin, kanamycin, chloramphenicol ortetracyclin resistance. Furthermore, the vector may comprise Aspergillusselection markers such as amdS, argB, niaD and sC, a marker giving riseto hygromycin resistance, or the selection may be accomplished byco-transformation, e.g., as described in WO 91/17243.

The procedures used to ligate the DNA construct of the inventionencoding a glucoamylase variant, the promoter, terminator and otherelements, respectively, and to insert them into suitable vectorscontaining the information necessary for replication, are well known topersons skilled in the art (cf., for instance, Sambrook et al.,Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor,1989).

Examples of suitable promoters for directing the transcription of theDNA sequence encoding an alpha-amylase variant of the invention,especially in a bacterial host, are the promoter of the lac operon of E.coli, the Streptomyces coelicolor agarase gene dagA promoters, thepromoters of the Bacillus licheniformis alpha-amylase gene (amyL), thepromoters of the Bacillus stearothermophilus maltogenic amylase gene(amyM), the promoters of the Bacillus amyloliquefaciens alpha-amylase(amyQ), the promoters of the Bacillus subtilis xylA and xylB genes etc.For transcription in a fungal host, non-limiting examples of usefulpromoters are those derived from the gene encoding A. oryzae TAKAamylase, the TPI (triose phosphate isomerase) promoter from S.cerevisiae (Alber et al., 1982, J. Mol. Appl. Genet. 1: 419-434,Rhizomucor miehei aspartic proteinase, A. niger neutral alpha-amylase,A. niger acid stable alpha-amylase, A. niger glucoamylase, Rhizomucormiehei lipase, A. oryzae alkaline protease, A. oryzae triose phosphateisomerase or A. nidulans acetamidase, and mutated, truncated, and/orhybrid promoters thereof.

Examples of preferred terminators for filamentous fungal host cells areobtained from the genes for Aspergillus oryzae TAKA amylase, Aspergillusniger glucoamylase, Aspergillus nidulans anthranilate synthase,Aspergillus niger alpha-glucosidase, and Fusarium oxysporum trypsin-likeprotease.

The control sequence may also be a suitable leader sequence, anontranslated region of an mRNA which is important for translation bythe host cell. The leader sequence is operably linked to the 5′ terminusof the nucleotide sequence encoding the polypeptide. Any leader sequencethat is functional in the host cell of choice may be used in the presentinvention.

Preferred leaders for filamentous fungal host cells are obtained fromthe genes for Aspergillus oryzae TAKA amylase and Aspergillus nidulanstriose phosphate isomerase.

The control sequence may also be a polyadenylation sequence, a sequenceoperably linked to the 3′ terminus of the nucleotide sequence and which,when transcribed, is recognized by the host cell as a signal to addpolyadenosine residues to transcribed mRNA. Any polyadenylation sequencewhich is functional in the host cell of choice may be used in thepresent invention.

Preferred polyadenylation sequences for filamentous fungal host cellsare obtained from the genes for Aspergillus oryzae TAKA amylase,Aspergillus niger glucoamylase, Aspergillus nidulans anthranilatesynthase, Fusarium oxysporum trypsin-like protease, and Aspergillusniger alpha-glucosidase.

The control sequence may also be a signal peptide coding region thatcodes for an amino acid sequence linked to the amino terminus of apolypeptide and directs the encoded polypeptide into the cell'ssecretory pathway. The 5′ end of the coding sequence of the nucleotidesequence may inherently contain a signal peptide coding region naturallylinked in translation reading frame with the segment of the codingregion which encodes the secreted polypeptide. Alternatively, the 5′ endof the coding sequence may contain a signal peptide coding region whichis foreign to the coding sequence. The foreign signal peptide codingregion may be required where the coding sequence does not naturallycontain a signal peptide coding region. Alternatively, the foreignsignal peptide coding region may simply replace the natural signalpeptide coding region in order to enhance secretion of the polypeptide.However, any signal peptide coding region which directs the expressedpolypeptide into the secretory pathway of a host cell of choice may beused in the present invention.

Effective signal peptide coding regions for bacterial host cells are thesignal peptide coding regions obtained from the genes for Bacillus NCIB11837 maltogenic amylase, Bacillus stearothermophilus alpha-amylase,Bacillus licheniformis subtilisin, Bacillus licheniformis alpha-amylase,Bacillus stearothermophilus neutral proteases (nprT, nprS, nprM), andBacillus subtilis prsA. Further signal peptides are described by Simonenand Palva, 1993, Microbiological Reviews 57: 109-137.

Effective signal peptide coding regions for filamentous fungal hostcells are the signal peptide coding regions obtained from the genes forAspergillus oryzae TAKA amylase, Aspergillus niger neutral amylase,Aspergillus niger glucoamylase, Rhizomucor miehei aspartic proteinase,Humicola insolens cellulase, and Humicola lanuginosa lipase.

The control sequence may also be a propeptide coding region that codesfor an amino acid sequence positioned at the amino terminus of apolypeptide. The resultant polypeptide is known as a proenzyme orpropolypeptide (or a zymogen in some cases). A propolypeptide isgenerally inactive and can be converted to a mature active polypeptideby catalytic or autocatalytic cleavage of the propeptide from thepropolypeptide. The propeptide coding region may be obtained from thegenes for Bacillus subtilis alkaline protease (aprE), Bacillus subtilisneutral protease (nprT), Saccharomyces cerevisiae alpha-factor,Rhizomucor miehei aspartic proteinase, and Myceliophthora thermophilalaccase (WO 95/33836). Where both signal peptide and propeptide regionsare present at the amino terminus of a polypeptide, the propeptideregion is positioned next to the amino terminus of a polypeptide and thesignal peptide region is positioned next to the amino terminus of thepropeptide region.

It may also be desirable to add regulatory sequences which allow theregulation of the expression of the polypeptide relative to the growthof the host cell. Examples of regulatory systems are those which causethe expression of the gene to be turned on or off in response to achemical or physical stimulus, including the presence of a regulatorycompound. Regulatory systems in prokaryotic systems include the lac,tac, and trp operator systems. In filamentous fungi, the TAKAalpha-amylase promoter, Aspergillus niger glucoamylase promoter, andAspergillus oryzae glucoamylase promoter may be used as regulatorysequences.

Other examples of regulatory sequences are those which allow for geneamplification. In eukaryotic systems, these include the dihydrofolatereductase gene which is amplified in the presence of methotrexate, andthe metallothionein genes which are amplified with heavy metals. Inthese cases, the nucleotide sequence encoding the polypeptide would beoperably linked with the regulatory sequence.

The vector may be an autonomously replicating vector, i.e., a vectorwhich exists as an extrachromosomal entity, the replication of which isindependent of chromosomal replication, e.g., a plasmid, anextrachromosomal element, a minichromosome, or an artificial chromosome.

The vector may contain any means for assuring self-replication.Alternatively, the vector may be one which, when introduced into thehost cell, is integrated into the genome and replicated together withthe chromosome(s) into which it has been integrated. Furthermore, asingle vector or plasmid or two or more vectors or plasmids whichtogether contain the total DNA to be introduced into the genome of thehost cell, or a transposon may be used.

The vectors of the present invention preferably contain one or moreselectable markers which permit easy selection of transformed cells. Aselectable marker is a gene the product of which provides for biocide orviral resistance, resistance to heavy metals, prototrophy to auxotrophs,and the like.

Examples of bacterial selectable markers are the dal genes from Bacillussubtilis or Bacillus licheniformis, or markers which confer antibioticresistance such as ampicillin, kanamycin, chloramphenicol ortetracycline resistance. Selectable markers for use in a filamentousfungal host cell include, but are not limited to, amdS (acetamidase),argB (ornithine carbamoyltransferase), bar (phosphinothricinacetyltransferase), hygB (hygromycin phosphotransferase), niaD (nitratereductase), pyrG (orotidine-5′-phosphate decarboxylase), sC (sulfateadenyltransferase), trpC (anthranilate synthase), as well as equivalentsthereof.

Preferred for use in an Aspergillus cell are the amdS and pyrG genes ofAspergillus nidulans or Aspergillus oryzae and the bar gene ofStreptomyces hygroscopicus.

The vectors of the present invention preferably contain an element(s)that permits stable integration of the vector into the host cell'sgenome or autonomous replication of the vector in the cell independentof the genome.

For integration into the host cell genome, the vector may rely on thenucleotide sequence encoding the polypeptide or any other element of thevector for stable integration of the vector into the genome byhomologous or nonhomologous recombination. Alternatively, the vector maycontain additional nucleotide sequences for directing integration byhomologous recombination into the genome of the host cell. Theadditional nucleotide sequences enable the vector to be integrated intothe host cell genome at a precise location(s) in the chromosome(s). Toincrease the likelihood of integration at a precise location, theintegrational elements should preferably contain a sufficient number ofnucleotides, such as 100 to 1,500 base pairs, preferably 400 to 1,500base pairs, and most preferably 800 to 1,500 base pairs, which arehighly homologous with the corresponding target sequence to enhance theprobability of homologous recombination. The integrational elements maybe any sequence that is homologous with the target sequence in thegenome of the host cell. Furthermore, the integrational elements may benon-encoding or encoding nucleotide sequences. On the other hand, thevector may be integrated into the genome of the host cell bynon-homologous recombination.

For autonomous replication, the vector may further comprise an origin ofreplication enabling the vector to replicate autonomously in the hostcell in question. Examples of bacterial origins of replication are theorigins of replication of plasmids pBR322, pUC19, pACYC177, and pACYC184permitting replication in E. coli, and pUB110, pE194, pTA1060, and pAMβ1permitting replication in Bacillus. An example of a sequence ensuringautonomous maintenance in a filamentous fungal host cell is the AMA1sequence. The origin of replication may be one having a mutation whichmakes its functioning temperature-sensitive in the host cell (see, e.g.,Ehrlich, 1978, Proceedings of the National Academy of Sciences USA 75:1433).

More than one copy of a nucleotide sequence of the present invention maybe inserted into the host cell to increase production of the geneproduct. An increase in the copy number of the nucleotide sequence canbe obtained by integrating at least one additional copy of the sequenceinto the host cell genome or by including an amplifiable selectablemarker gene with the nucleotide sequence where cells containingamplified copies of the selectable marker gene, and thereby additionalcopies of the nucleotide sequence, can be selected for by cultivatingthe cells in the presence of the appropriate selectable agent.

Host Cells

The cell of the invention, either comprising a DNA construct or anexpression vector of the invention as defined above, is advantageouslyused as a host cell in the recombinant production of an alpha-amylasevariant of the invention. The cell may be transformed with the DNAconstruct of the invention encoding the variant, conveniently byintegrating the DNA construct (in one or more copies) in the hostchromosome. This integration is generally considered to be an advantageas the DNA sequence is more likely to be stably maintained in the cell.Integration of the DNA constructs into the host chromosome may beperformed according to conventional methods, e.g., by homologous orheterologous recombination. Alternatively, the cell may be transformedwith an expression vector as described above in connection with thedifferent types of host cells.

The cell of the invention may be a cell of a higher organism such as amammal or an insect, but is preferably a microbial cell, e.g., abacterial or a fungal (including yeast) cell.

Examples of suitable bacteria are Gram-positive bacteria such asBacillus subtilis, Bacillus licheniformis, Bacillus lentus, Bacillusbrevis, Bacillus stearothermophilus, Bacillus alkalophilus, Bacillusamyloliquefaciens, Bacillus coagulans, Bacillus circulans, Bacilluslautus, Bacillus megaterium, Bacillus thuringiensis, or Streptomyceslividans or Streptomyces murinus, or gram-negative bacteria such as E.coli. The transformation of the bacteria may, for instance, be effectedby protoplast transformation or by using competent cells in a mannerknown per se.

The host cell may also be a filamentous fungus, e.g., a strain belongingto a species of Aspergillus, most preferably Aspergillus oryzae orAspergillus niger, or a strain of Fusarium, such as a strain of Fusariumoxysporium, Fusarium graminearum (in the perfect state named Gribberellazeae, previously Sphaeria zeae, synonym with Gibberella roseum andGibberella roseum f. sp. cerealis), or Fusarium sulphureum (in theprefect state named Gibberella puricaris, synonym with Fusariumtrichothecioides, Fusarium bactridioides, Fusarium sambucium, Fusariumroseum, and Fusarium roseum var. graminearum), Fusarium cerealis(synonym with Fusarium crokkwellnse), or Fusarium venenatum.

In a preferred embodiment of the invention the host cell is a proteasedeficient or protease minus strain. This may for instance be theprotease deficient strain of the genus Aspergillus, in particular astrain of A. oryzae, such as A. oryzae JaL125 having the alkalineprotease gene named “alp” deleted. This strain is described in WO97/35956 (Novo Nordisk).

Filamentous fungi cells may be transformed by a process involvingprotoplast formation and transformation of the protoplasts followed byregeneration of the cell wall in a manner known per se. The use ofAspergillus as a host micro-organism is described in EP 0238023 (NovoNordisk), the contents of which are hereby incorporated by reference.

An aspect of the invention relates to a recombinant host cell comprisinga nucleic acid construct as defined above, or at least one copy of anexpression vector as defined above.

A preferred embodiment relates to a cell of the previous aspect, whichis a microorganism; preferably a cell which is a bacterium or a fungus;more preferably a cell which is a gram-positive bacterium such asBacillus subtilis, Bacillus licheniformis, Bacillus lentus, Bacillusbrevis, Bacillus stearothermophilus, Bacillus alkalophilus, Bacillusamyloliquefaciens, Bacillus coagulans, Bacillus circulans, Bacilluslautus or Bacillus thuringiensis; or most preferably a cell which is aprotease deficient strain of the fungus Aspergillus, in particular A.oryzae.

Method of Producing an Alpha-Amylase Variant of the Invention

In a yet further aspect, the present invention relates to a method ofproducing an alpha-amylase variant of the invention, which methodcomprises cultivating a host cell under conditions conducive to theproduction of the variant and recovering the variant from the cellsand/or culture medium.

The medium used to cultivate the cells may be any conventional mediumsuitable for growing the host cell in question and obtaining expressionof the alpha-amylase variant of the invention. Suitable media areavailable from commercial suppliers or may be prepared according topublished recipes (e.g., as described in catalogues of the American TypeCulture Collection).

The alpha-amylase variant secreted from the host cells may convenientlybe recovered from the culture medium by well-known procedures, includingseparating the cells from the medium by centrifugation or filtration,and precipitating proteinaceous components of the medium by means of asalt such as ammonium sulphate, followed by the use of chromatographicprocedures such as ion exchange chromatography, affinity chromatography,or the like.

Various methods for using such produced amylases, as well as morespecific uses are outlined in other aspects of the invention as alreadymentioned in the summary of this invention.

The present invention provides a method of using alpha-amylase encodedby the polynucleotides of the invention for producing glucose or maltoseor the like from starch.

Generally, the method includes the steps of partially hydrolyzingprecursor starch in the presence of alpha-amylase and then furtherhydrolyzing the release of D-glucose from the non-reducing ends of thestarch or related oligo- and polysaccharide molecules in the presence ofglucoamylase by cleaving alpha-1,4 and alpha-1,6 glucosidic bonds.

The partial hydrolysis of the precursor starch utilizing alpha-amylaseprovides an initial breakdown of the starch molecules by hydrolyzinginternal alpha-(1,4)-linkages. In commercial applications, the initialhydrolysis using alpha-amylase is run at a temperature of approximately105° C. A very high starch concentration is processed, usually 30% to40% dry-solids. The initial hydrolysis is usually carried out forapprox. five minutes at this elevated temperature. The partiallyhydrolyzed starch can then be transferred to a second tank and incubatedfor approximately one hour at a temperature of 85° to 90° C. to derive adextrose equivalent (D.E.) of 10 to 15.

The step of further hydrolyzing the release of D-glucose from thenon-reducing ends of the starch or related oligo- and polysaccharidesmolecules in the presence of glucoamylase is normally carried out in aseparate tank at a reduced temperature between 30 and 60° C. Preferablythe temperature of the substrate liquid is dropped to between 55 and 60°C. The pH of the solution is dropped from 6-6.5 to a range between 3 and5.5. Preferably, the pH of the solution is 4 to 4.5. The glucoamylase isadded to the solution and the reaction is carried out for 24-72 hours,preferably 36-48 hours.

The alpha-amylases encoded by the polynucleotides of the invention mayalso be used in brewing processes. Further, the alpha-amylase encoded bythe polynucleotides of the invention may be used for maltose production.High maltose syrup is typically produced as follows.

To produce “High Maltose Syrup” (containing 50-55% maltose), starch isliquefied to DE 10-20. The pH and temperature of the liquefied starch isadjusted to 65° C. and to a pH around 5.0, respectively, and issubjected to maltogenic alpha-amylase activity (e.g., Bacillusstearothermophilus amylase, such as Maltogenase™ 4000 L, 0.4 l/t DS(Novozymes)), pullulanase activity (e.g., Bacillus pullulanase, such asPromozyme™ 600 L, 0.3 l/t DS (Novozymes)) and alpha-amylase activity(e.g., BAN 240 L or Termamyl™ 120 L, type LS, 0.4 kg/t DS (Novozymes))for 24-41 hours. The specific process time depends on the desiredsaccharide spectrum to be achieved. By increasing the dosage of themaltogenic alpha-amylase and pullulanase the maltose content can beincreased.

Alternatively, “High Maltose Syrup” may be produced by first liquefyingstarch to DE 10-20 and then adjusting the pH and temperature to 55° C.or higher and a pH around 5.5 or lower, and then subjecting theliquefied starch to a fungal alpha-amylase activity (e.g., Bacillusstearothermophilus amylase, such as Fungamyl™ 800L (Novozymes)) for22-44 hours. The dosage of fungal Fungamyl™ 800L depends on thesaccharification time foreseen, e.g., 200 g/t DS for 44 hours and 400g/t DS for 22 hours. The alpha-amylases encoded by the polynucleotidesof the invention may substitute the Fungamyl™ 800L in the above process,and then the temperature can be even higher, and the pH even lower,resulting in a faster conversion rate, and thus a better overalleconomy.

To produce “High Maltose Syrup” starch with maltose content of 55-65%starch is liquefied to DE 10-20. The temperature and pH of the liquefiedstarch is adjusted to 60° C. or higher, and to a pH around 6 or lower,and is subjected to maltogenic alpha-amylase activity (e.g.,Maltogenase™ 4000 L, 0.25-1.0 l/t DS (Novozymes)), and fungalalpha-amylase activity (e.g., Aspergillus amylase, such as Fungamyl™ 800L, 0.4-1.0 kg/t DS (Novo Nordisk) for 24-48 hours; or the alpha-amylaseencoded by the polynucleotide of the invention for a shorter time.

The alpha-amylase variant of the invention may also be used in bakingprocesses. In one aspect the invention relates to the used of a variantof the invention for starch conversion, alcohol production, brewing, andbaking.

The invention also relates to a process of producing maltose syrupcomprising the steps of: 1) liquefying starch in the presence of analpha-amylase; 2) dextrinization in the presence of a fungalalpha-amylase variant of the invention; and 3) recovery of the syrup;and optional purification of the syrup.

The alpha-amylase used for liquefaction in step 1) may be anyalpha-amylase. Preferred alpha-amylase are Bacillus alpha-amylases, suchas a Termamyl-like alpha-amylase, which including the B. licheniformisalpha-amylase (commercially available as Termamyl™ (Novo Nordisk)), theB. amyloliquefaciens alpha-amylase (sold as BAN (Novo Nordisk), the B.stearothermophilus alpha-amylase (sold as Termamyl™ 120 L type S), Thealpha-amylases derived from a strain of the Bacillus sp. NCIB 12289,NCIB 12512, NCIB 12513 or DSM 9375, all of which are described in detailin WO 95/26397, and the alpha-amylase described by Tsukamoto et al.,1988, Biochemical and Biophysical Research Communications, 151: 25-31.Alpha-amylases within the definition of “Termamyl-like alpha-amylase”are defined in for instance WO 96/23874 (Novo Nordisk).

In another aspect the invention relates to a process of producingmaltose comprising the steps of: 1) liquefying starch at a temperatureof 140-160° C. at a pH of 4-6; 2) dextrinization at a temperature in therange from 60-95° C., in particular at 65-85° C., such as 70-80° C., ata pH 4-6 in the presence of a fungal alpha-amylase variant of theinvention; and 3) recovery of the syrup; and optional purification ofthe syrup.

In an embodiment of the invention an effective amount of glucoamylase isadded in step 2). The syrup will in this embodiment (including treatmentwith a glucoamylase) not be maltose syrup, but syrup with a differentsugar profile. The glucoamylase may be an Aspergillus glucoamylase, inparticular an Aspergillus niger glucoamylase.

Alternatively, the process comprising the steps of: 1) liquefying starchat a temperature of 95-110° C. at a pH of 4-6 in the presence of aBacillus alpha-amylase; 2) liquefying at a temperature in the range from70-95° C. at a pH 4-6 in the presence of an alpha-amylase encoded by apolynucleotide as defined in the first aspect of the invention, followedby recovery and/or optional purification of the product obtained.

Finally, some aspects of the invention relate to various detergent uses.One aspect relates to a detergent additive comprising an alpha-amylaseencoded by a polynucleotide as defined in the first aspect, optionallyin the form of a non-dusting granulate, stabilized liquid or protectedenzyme. A preferred embodiment of this aspect relates to a detergentadditive which contains 0.02-200 mg of enzyme protein/g of the additive.Another preferred embodiment relates to a detergent additive accordingto the previous aspect, which additionally comprises another enzyme suchas a protease, a lipase, a peroxidase, another amylolytic enzyme and/ora cellulase. Another aspect relates to a detergent compositioncomprising an alpha-amylase encoded by a polynucleotide as defined inthe first aspect, and a preferred embodiment of this aspect relates to adetergent composition which additionally comprises another enzyme suchas a protease, a lipase, a peroxidase, another amylolytic enzyme and/ora cellulase. Still another aspect relates to a manual or automaticdishwashing detergent composition comprising an alpha-amylase variantencoded by a polynucleotide as defined in the first aspect. A preferreddishwashing detergent composition additionally comprises another enzymesuch as a protease, a lipase, a peroxidase, another amylolytic enzymeand/or a cellulase. A final detergent related aspect is a manual orautomatic laundry washing composition comprising an alpha-amylasevariant encoded by a polynucleotide as defined in the first aspect; anda preferred laundry washing composition according additionally comprisesanother enzyme such as a protease, a lipase, a peroxidase, an amylolyticenzyme and/or a cellulase.

EXAMPLES Example 1 Purification and Characterization of theAlpha-Amylase from Rhizomucor pusillus NN046782

This alpha-amylase denoted AM782 was purified from culture broth ofthermophilic fungal strain NN046782, and it was found to be more stablethan the BAN (Bacillus amyloliquefaciens) amylase at 60, 70, and 80° C.,at pH=5.0, 6.0, and 7.0. The characteristics are summarized asfollowing:

Molecular weight (SDS) ≈50 kDa (SDS-PAGE) pI pH 3.5 Active pH range pH3-9 Optimal pH pH 4-5 Active temperature range 30-80° C. OptimalTemperature 70° C. pH Stability stable at pH = 5, 6, 7.

Media for Fungal Growth

YG: Yeast-glucose agar 5.0 g Difco powdered yeast extract 10.0 g glucose20.0 g agar 1000 ml tap water Autoclave at 121° C. for 15-20 min.

FG-4 Media 50 ml/flask: 30 g Soymeal, 15 g Maltose 5 g Peptone, 1000 mlH₂O 1 g olive oil (2 drops/flask) 50 ml in 500 ml Erlenmeyer flask with2 baffles. Autoclave at 121° C. for 30 min.

The fungi were grown on YG agar plate (4.5 cm diameter) for 3 days under45° C. in the darkness and used for inoculating shake flask. The plateswith fully grown cultures were stored at 4° C. before use.

For enzyme production, 4-6 agar plugs with fully grown fungal cultureson the above plates were used to inoculate one shake flask with FG-4 andgrown under 45° C., 160 rpm for 72 hours, then harvested by centrifugedthe culture broth at 8000 rpm and 4° C. for 30 minutes. The supernatantwas collected and used for enzyme purification.

Chemicals and Regeants

BAN standard and Fungamyl™ 800L (Novozymes A/S, Denmark) were used asbenchmark. AZCL-amylose (Megazyme) was used for enzyme assay.

Other Chemical and Buffers Include:

25 mM Tris-HCl, pH 7.0; 25 mM Tris-HCl; 1 M NaCl, pH7.0; 0.1 MNa₃PO₄/Citric Acid, pH 5.5; ammonium sulfate; 0.1 M NaAc, pH 5.0; 0.1 MMES, pH 6.0; 0.1 M Tris-HCl, pH 7.0

Buffer for pH profile: 100 mM succinic acid, 100 mM HEPES, 100 mM CHES,100 mM CABS, 1 mM CaCl₂, 150 mM KCl, 0.01% Triton X-100 adjusted topH-values 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0 and 11.0 with HClor NaOH

Enzyme Activity Tests Microtiter Plate Assay:

The supernatants were tested for alpha-amylase activity by microtiterplate assay. A solution of 0.2% of the blue substrate AZCL-amylose(Megazyme) was suspended in a 0.1 M phosphate-citrate buffer (pH 5.5) orTris-HCl buffer (pH 7) under stirring. The solution was distributedunder stirring to a microtiter plate (200 microliters to each well), 20microliters enzyme sample was added and the plates were incubated in anEppendorf Thermomixer for 15-30 minutes at 50° C. and 650 rpm. Denaturedenzyme sample was prepared at 100° C. boiling for 20 min and then usedas blank controls. After incubation the colored solution was separatedfrom the solid by centrifugation at 3000 rpm for 5 minutes at 4° C. Then150 microliters of supernatant was transferred to a microtiter plate andthe absorbance was measured in a BioRad Microplate Reader at 595 nm.

Eppendorf Tube Assay:

A solution of 0.2% of the blue substrate AZCL-amylose (Megazyme) wassuspended in buffers at different pH-values under stirring. Thesolutions were distributed under stirring to 1.5 ml Eppendorf tubes (900microliters to each), 100 micro-m enzyme sample is added to each tubeand they were then incubated in a waterbath for 10-60 min. at 50° C.Denatured enzyme samples (prepared by 100° C. boiling for 20 min) wereused as blank controls. After incubation the colored solution wasseparated from the solid by centrifugation at 5000 rpm for 10 minutes at4° C. Then 200 microliters of supernatant was transferred into amicrotiter plate and the absorbence was measured in a BioRad MicroplateReader at 595 nm.

Isoelectric Focusing

Isoelectric focusing was carried out in precast Apholine PAG plate pH3.5-9.5 (Pharmacia, Sweden) according to the manufacturer'sinstructions. The samples were applied in triplicate and afterelectrophoresis the gel was divided into three. An overlay containing 1%agarose and 0.4% AZCL-amylose in buffer pH 5-7 was poured onto each partof the gel which was incubated at 45° C. for 12-16 hours. Enzymeactivity and pl of enzyme protein was identified by blue zones.

SDS-PAGE

For checking of purifity and determining the molecular weight ofpurified amylase, 30□ microliters of enzyme samples were applied to 12%SDS-poly acrylamide gel electrophoresis. The gel was run at 100 V for1.5 hrs and stained with Coomassie blue.

Enzyme Purification

300 ml supernatant of the strain NN046782 was precipitated with ammoniumsulfate (80% saturation) and redissolved in 20 ml 25 mM Tris-HCl buffer,pH7.0, then dialyzed against the same buffer and filtered through a 0.45mm filter, the final volume was 200 ml. The solution was applied to a 35ml Source 15Q column (Phamacia) equilibrated in 25 mM Tris-HCl buffer,pH 7.0, and the proteins was eluted with a linear NaCl gradient (0-0.3M). Fractions from the column were analyzed for amylase activity onAZCL-amylose at pH 5.5. Fractions with amylase activity were pooled.Then the pooled solution was ultrafiltrated, the concentrated solutionwas applied to a 180 ml Superdex75 column equilibrated with 25 mMTris-HCl, pH7.0, the proteins was eluted with the same buffer. Amylasecontaining fractions were analyzed by SDS-PAGE and pure fractions werepooled.

Enzyme Characterization pH Profile:

20 microliters enzyme sample and 200 microliters 0.2% AZCL-amylose inthe following buffer system (100 mM succinic acid, 100 mM HEPES, 100 mMCHES, 100 mM CABS, 1 mM CaCl₂, 150 mM KCl, 0.01% Triton X-100 adjustedto pH-values 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0 and 11.0 withHCl or NaOH) with different pH were mixed in an Microtiter plate andplaced on ice before reaction. The assay was initiated by transferringthe Microtiter plate to an Eppendorf thermomixer, which was set to theassay temperature 50° C. The plate was incubated for 20 minutes on theEppendorf thermomixer at 650 rpm shaking rate. The incubation wasstopped by transferring the plate back to the ice bath. Then the platewas centrifuged in an ice-cold centrifuge for a few minutes and 150microliters supernatant was transferred to a new microtiter plate. Theabsorbance, OD₅₉₅, was read as a measure of amylase activity. Allreactions were done in triplicate, and a buffer blind was included inthe assay (instead of enzyme). BAN and Fungamyl™ commercial enzymes wereused as positive controls. The results are shown in FIG. 1.

Temperature Profile:

Eppendorf tubes with 200 microliters 0.2% AZCL-amylose in 0.1 MNa₃PO₄/Citric acid buffer pH 5.5 were pre-incubated at 20, 30, 40, 50,55, 60, 70, 80° C. The assay was initiated by mixing 20 microlitersenzyme sample with the buffer. The tubes were incubated for 10 minuteson the Eppendorf thermomixer at its highest shaking rate (1400 rpm). Theincubation was stopped by transferring the tube to the ice bath. Thenthe tubes were centrifuged in an icecold centrifuge for a few minutesand 150 microliters supernatant was transferred to a microtiter plate.OD₅₉₅ was read as a measure of amylase activity. All reaction was donewith triplicate and a buffer blind was included in the assay (instead ofenzyme). BAN and Fungamyl™ were used as control. The results are shownin FIG. 2.

pH and Themperature Stability:

80 microliters enzyme sample (diluted with 0.1 M NaAc pH 5.0, 0.1 M MESpH 6.0, 0.1 M Tris-HCl pH7.0 respectively) in an Eppendorf tube wasincubated for 5, 10, 15 and 20 minutes on the Eppendorf Thermomixer at60, 70, 80° C. and 300 rpm shaking. The incubation was stopped bytransferring the tube back to the ice bath. Un-incubated sample was usedas control. The 20 microliters of the above incubated sample wastransferred into a new microtiter plate and 200 microliters 0.2%AZCL-amylose in 0.1 M Na₃PO₄/Citric buffer pH 5.5 was added. The assaywas initiated by transferring the Microtiter plate to an Eppendorfthermomixer, which was set to the assay temperature 50° C. The plate wasincubated for 30 minutes on the Eppendorf thermomixer at 650 rpm shakingrate. The incubation was stopped by transferring the plate back to theice bath. Then the plate was centrifuged in an ice-cold centrifuge for afew minutes and 150 microliters supernatant was transferred to a newmicrotiter plate. OD₅₉₅ was read as a measure of amylase activity. Allreaction was done with duplicate and a buffer blind was included in theassay (instead of enzyme). BAN and Fungamyl™ were used as control. Theresults are shown in FIGS. 3-5.

Side Activity Assay:

The purified amylase was tested in follow substrates at pH 7.0:AZCL-galactomannan, AZCL-beta-glucan, AZCL-dextran, AZCL-xyloglucan,AZCL-potato galactan, AZCL-arabinan, AZCL-pullulan, AZCL-xylan,AZCL-he-cellulose, AZCL-casein. No side-activity was detected in any ofthe substrates.

The purity of purified amylase was checked in 12% SDS-page, themolecular weight of the enzyme is around 50 KDa as seen on SDS-PAGE, thepl of AM782 is around pH3.5 as determined by IEF.

Example 2 Cloning of the Gene Encoding the AM782 Alpha-Amylase ofRhizomucor pusillus NN046782 Fungal Strain and its Growth

Rhizomucor pusillus NN046782 was grown at 45° C., 165 rpm for 48 hoursin FG4 medium with 2% starch. The mycelium was harvested bycentrifugation at 7000 rpm for 30 minutes. The harvested mycelium wasstored at −80° C. before use for RNA extraction.

Extraction of Total RNA

Total RNA was extracted from 100 mg mycelium using the RNeasy Mini Kit(Qiagen).

Specific Primers

It was found that the amylase AM782 from Rhizomucor pusillus NN046782had the same N-terminal sequence as an amylase encoding gene which hadbeen identified and sequenced by us at in an earlier project fromanother Rhizomucor pusillus strain NN101459. Thus two specific primerswere designed from the previously determined DNA sequence, and thesewere used for the cloning of amylase from NN046782.

Primer AM298-CDSF (SEQ ID NO: 1): 5′-tat cat gaa att cag catPrimer AM298-CDSR (SEQ ID NO: 2): 5′-agt tca aaa tgg aca aag t

The following PCR reaction system and conditions were used:

Pfu DNA polymerase 10 x PCR buffer with MgSO₄ 5 microliters 10 mM dNTPmix 1 microliter Primer AM298-CDSF (10 micro-M) 1 microliter PrimerAM298-CDSR (10 micro-M) 1 microliter pfu DMA polymerase (3 u/microliter)0.5 microliter cDNA synthesis reaction (template) 2 microliters Addautoclaved, distilled water to 50 microliters

Conditions:

95° C. 3 min 95° C. 30 sec 45 or 50 or 53° C. 30 sec 40 cycles 72° C. 3min 72° C. 7 min

The PCR product was viewed on agarose gel and a specific band wasidentified and purified. Thus 0.3 microliter of this PCR product wasused as template for second round PCR at the following conditions.

Pfu DNA polymerase 10 x PCR buffer with MgSO₄ 5 microliters 10 mM dNTPmix 1 microliter Primer AM298-CDSF (10 micro-M) 1 microliter PrimerAM298-CDSR (10 micro-M) 1 microliter pfu DNA polymerase (3 u/microliter)0.5 microliter cDNA synthesis reaction 0.3 microliter Add autoclaved,distilled water to 50 microliters

Conditions:

95° C. 3 min 95° C. 30 sec 55° C. 30 sec 40 cycles 72° C. 3 min 72° C. 7min

A specific band with the size of about 1.5 kb was the result of thisamplification. A polyA tail was added using Taq DNA polymerase (PCRproduct 20 microliters, 10× buffer 2 microliters, Mg2+1 microliter, dATP(10 mM) 0.5 microliter, Taq polymerase (5 unit/microliter) 0.3microliter) and incubation at 72° C. for 30 min. The dA-tailed fragmentwas recovered from the gel with GFX Kit and redissolve into 30microliters water. Then the purified fragment was ligated into thepGEM-T Vector (by mixing 2× buffer 10 microliters, T-vector (50 ng/l) 1microliter, T4 ligase (3 unit/I) 1 microliter and purified PCR product 8microliters; then let it stay overnight at 4° C.), and transformed itinto the competent cells (transformation condition: 1 microliterligation solution and 40 microliters DH10B competent cell in 0.1 cmcurvette, 1.8 KV). 8 positive clones were screened by colony PCR.

Colony PCR System:

10 x PCR buffer 5 microliters 25 mM MgCl₂ 3 microliters 10 mM dNTP mix 1microliter AM298-CDSF 1 microliter AM298-CDSR 1 microliter pfupolymerase 1 microliter Add autoclaved, distilled water to 50microliters

A white colony was transferred directly into PCR mixture where it servedas the template. The PCR conditions were as follows:

94° C. 3 min 94° C. 30 sec 55° C. 30 sec 30 cycles 72° C. 1 min 72° C.10 min

After PCR reaction, 10 microliters PCR products were loaded into 1%agarose in 0.5×TBE buffer and run through the gel under 90 V for 1 hourand then visualized under UV, all colonies gave positive result.

Then plasmid was extracted from 3 of these 8 clones with Wizards PlusMinipreps DNA Purification System (Promega). The plasmids were sequencedusing the ET terminater kit (Amersham) with the two primers AM298-CDSFand AM298-CDFR, and the 3 clones turned out to be identical, thefull-length sequence is shown in SEQ ID NO: 3.

A plasmid comprising a DNA sequence encoding the alpha-amylase AM782 hasbeen transformed into a strain of the Escherichia coli DH10B which wasdeposited by the inventors according to the Budapest Treaty on theInternational Recognition of the Deposit of Microorganisms for thePurposes of Patent Procedure at the Deutsche Sammlung vonMikroorganismen and Zellkulturen GmbH, Mascheroder Weg 1b, D-38124Braunschweig, Federal Republic of Germany, on 29 Nov. 2002 under thedeposition number DSM 15334. The deposit was made by Novozymes A/S. Itis contemplated that the DNA sequence of this plasmid comprises the DNAsequence of SEQ ID NO: 3.

Further sequence analysis of the cDNA clone showed that the sequencecontains a coding region of 1413 nucleotides. The translation product ofthe coding region is a peptide of 471 amino acids. The deduced aminoacid sequence encoded by this gene, with a signal peptide (from aa 1-21)and a mature peptide (from aa 22-471) is shown in SEQ ID NO: 4.

Example 3 Subcloning and Heterologous Expression of AM782 AmylaseStrains and Plasmids

The A. oryzae strain BECh2, used as expression host has the followinggenotype: amy⁻, alp⁻, Npl⁻, CPA⁻, KA⁻ . E. coli DH5alpha (Invitrogen™)was used as cloning host in construction of the expression vector. Theexpression plasmid pDAu71 (FIG. 6) containing the A. nidulans amdS geneas selection marker in Aspergillus and the Ampicillin resistance genefor selection in E. coli, two copies of the A. niger NA2 promoter(neutral amylase) with 3 extra amyR-sites+the 5′ untranslated part ofthe A. nidulans TPI promoter for heterologous expression and the A.niger AMG terminator was used.

PCR Amplification:

10 x PCR buffer (incl. MgCl₂) 5 microliters 2.5 mM dNTP mix 5microliters 168/R.p. amy3-forw (10 μM) 5 microliters 169/R.p. amy4-rev(10 μM) 5 microliters Expand High Fidelity polymerase (Roche) 0.5microliter Template DNA 1 microliter Add autoclaved, distilled water to50 microliters

Conditions

95° C. 1 min 1 cycle 94° C. 30 sec 60° C. 30 sec 20 cycles 72° C. 1.30min 72° C. 2 min 1 cycle

Construction of plasmid pPFJo143 (FIG. 13): A DNA fragment containingthe AM782 gene was PCR amplified from a plasmid containing the fulllength cDNA with primers designed from the full sequence:

Primer 168/R.p. amy3-forw (SEQ ID NO: 5):gaagatctaccatgaaattcagcatctctctcPrimer 169/R.p. amy4-rev (SEQ ID NO: 6): ccgctcgagttaagcagaggtgaagatagc

The primers have cloning restriction sites BglII-XhoI, respectively, inthe ends. A pool of PCR product from individual PCR reactions was usedfor the cloning. The PCR product was digested with BglII and XhoI andcloned into pDAu71, digested with BamHI and XhoI. The PCR product wassequenced and verified to be identical to the original sequence.

Transformation of BECh2 was performed by a method involving protoplastformation and transformation of these. Suitable procedures forAspergillus transformation are described in EP 0238023 and Yelton etal., 1984, Proceedings of the National Academy of Sciences USA 81:1470-1474. Transformants were isolated and grown in smallNunc-containers in 10 ml of YPM (1% yeast extract, 2% Bacto peptone, and2% maltose) for 3 days at 30° C. (rotated).

SDS-page gel electrophoresis: 10 microliters supernatant samples fromthe above described 10 ml cultures were subjected to SDS-gelelectrophoresis. Gels were stained with SYPRO Orange Protein Gel Stain(Molecular Probes).

Alpha-amylase was assayed by PNP as follows. A working solution wasmade: 5 ml alpha-glucosidase solution+1 ml substrate solution(PNP-substrate). Termamyl™ was used as standard (concentrations from0-100 NU/ml). Buffer for dilution: 50 mM acetic acid, boric acid andphosphorous acid and 0.1 mM CaCl₂+0.25% BRIJ35. 20 microliterssupernatant in a microtiter plate was incubated for 2 mins. 200microliters working solution was added. Kinetics at 405 nm over 3 min.

The PCR amplified ORF was cloned into the expression vector pDAu71,resulting in pPFJo143 as described in Materials and Methods (FIG. 7).The plasmid was transformed into BECh2 (A. oryzae). 10 transformantswere isolated, grown in YPM for 3 days and supernatants run on anSDS-PAGE. This showed varying expression levels ranging from very littleto quite good expression. The molecular weight is around 50 kDa, inaccordance with what was found for the wild type enzyme. TransformantBECh2/pPFJo143-9 was chosen to ferment for a small scale purification ofthe amylase. An alpha-amylase assay was performed according to Materialsand Methods (Table 1). It was performed with Termamyl™ as a standard—andthe units are NU/ml—which means that it only gives us relative numbers,but we could see that there was a good correlation between activity andamount of protein.

TABLE 1 Supernatants from fermentation of the 10 transformants 143-1 to143-10 were subjected to an alpha-amylase assay using a PNP- substrate.The numbers are relative. Transf. 1 2 3 4 5 6 7 8 9 10 NU/ml 155 192 241195 166 240 107 212 342 205

Example 4 Investigation of the Sugar Profile of Maltodextrin Hydrolysisby the Alpha-Amylase AM782 Apparatus

Thermostated water bath Heto lab equipment DT1; pH-meter (MettlerMP220); HPLC Waters system (NP TSCN-QI-2411); Electronic pipette;Spectrophotometer UV-1601; Balance (Mettler AG 204); Refractometer.

Glassware

250 ml flask with lid; heavy rings for flask; 500 ml volumetric flask;500 ml beaker; 10 ml glass tubes.

Enzymes

AM 782 alpha-amylase at 0.225 FNU/ml, and as control a commerciallyavailable fungal amylase Fungamyl™ 800L (Novozymes A/S, Denmark;AFN-000515) at 0.135FNU/g.ds.

Substrate

DE 11 maltodextrin FFS-99039

Protocol

Two water baths were set up with a temperature of 55° C. and 70° C.respectively. 53 g of maltodextrin was slowly added into 397 g boilingMilli-Q water in a glass beaker and stirred with an electronic stirrersimultaneously, until all the maltodextrin was dissolved in the water.The weight of the maltodextrin solution was noted, and more boilingwater was added up to 450 g. The maltodextrin solution was transferredinto the water bath and cooled down to hydrolysis temperature. Then themaltodextrin solution was divided into two equal portions: one portionwas adjusted to pH 5.0 with 1 N HCl; the rest was kept at its natural pH(5.5). The substrate concentration was checked by refractometer (DSabout 10%).

The maltodextrin solutions were transferred into four 250 ml flasks withlids: flask #1 with 90 g pH 5.5 solution; flasks #2 & #3 with 90 g pH5.0 solution; flask #4 with 95 g pH 5.5 solution. The flasks were keptin the water baths at hydrolysis temperature, flasks #1-3 at 70° C., andflask #4 at 55° C.

10 ml diluted AM782 enzyme (6 ml AM782 sample with 4 ml Milli-Q water)was added to each of the flasks #1-3 containing 90 g solution, and 5 mldiluted Fungamyl™ (0.04 g Fungamyl in 100 ml Milli-Q water) was added toflask #4 containing 95 g solution. In this way, the AM782 and Fungamyl™was dosaged at the same activity level i.e., 0.135 FNU/g.ds.

The pH of each flask was checked and the DS of each flask was measuredby sampling at T=0 hours. Thereafter 4 ml samples were taken from eachshake flask at T=3, 6, 9, 21, 24, 28, 45, and 48 hours after incubationin the water baths.

The enzymes in the hydrolysis samples were inactivated immediately aftersampling by boiling the samples for 15 minutes, and then cooling themdown to room temperature for HPLC analysis. After cooling, pH and DS wasmeasured for each sample in order to determine sample dilution, and thenthe sample was diluted with Milli-Q water to a concentration of DS=5%.The samples were mixed with mixed bed ion-exchange resin (Bio-Rad AG501/X8 (D)) and left to stand for 20 minutes, this removed ash andsoluble N from the samples. The samples were then filtered through a 0.2micro-m filter (Sartorius MINISART™ NML 0.2 micron) and the filteredsamples were collected in HPLC bottles and analyzed by HPLC. The resultsare given in tables 2-4 below, and in FIG. 8.

TABLE 2 Flask #1 with AM782 (0.135 FAU/g.ds.) at 70° C., and initial pH5.5. Hours PH % DS % DP1 % DP2 % DP3 % DP4 3 6.05 7.59 52.63 16.54 23.256 6.03 13.74 57.39 8.61 20.26 9 6.02 16.46 57.78 6.12 19.64 21 5.8819.08 58.57 4.37 17.98 24 5.83 18.78 58.65 4.34 18.22 28 5.82 18.4758.92 4.25 18.37 45 5.61 19.04 58.71 4.07 18.18 48 5.58 12.3 18.72 58.594.28 18.42

TABLE 3 Flasks #2 & #3 with AM782 (0.135 FAU/g.ds.) at 70° C. andinitial pH 5.0. Hours PH % DS % DP1 % DP2 % DP3 % DP4 3 5.89 7.34 51.7416.56 24.37 6 5.88 13.66 56.96 8.61 20.78 9 5.87 16.13 57.29 6.53 20.0521 5.75 18.13 58.12 4.87 18.88 24 5.72 17.99 58.33 5.02 18.68 28 5.7417.54 58.69 4.83 18.95 45 5.53 17.79 58.45 4.87 18.90 48 5.52 11.9 17.8558.29 4.91 18.95

TABLE 4 Flask #4 with Fungamyl ™ 800L (0.135 FAU/g.ds.) at 55° C. andinitial pH 5.5. Hours PH % DS % DP1 % DP2 % DP3 % DP4 20 6.12 10.8 1.4145.27 28.19 25.13 24 6.05 10.9 1.73 45.39 27.67 25.21

This experiment showed that the amylase AM782 can work at a very hightemperature, at least up to 70° C. The amylase AM782 has a very fastreaction speed; when compared at the same dosage with Fungamyl™ 800 L,the amylase AM782 can achieve in about 3 hours, what takes Fungamyl™ 24to 48 hours. Furthermore, the amylase AM782 can degrade DP3 into DP2 andDP1, so it gives a higher DP1 result.

1. An isolated polynucleotide comprising an open reading frame encodinga polypeptide having alpha-amylase activity, the polypeptide selectedfrom the group consisting of: a) a polypeptide comprising an amino acidsequence which has at least 70% identity with amino acids 22 to 450 ofSEQ ID NO: 4; b) a polypeptide comprising an amino acid sequence whichhas at least 70% identity with the polypeptide encoded by the amylaseencoding part of the polynucleotide inserted into a plasmid present inthe E. coli host deposited under the Budapest Treaty with DSMZ underaccession number DSM 15334; c) a polypeptide encoded by a polynucleotidecomprising a nucleotide sequence which has at least 70% identity withthe sequence shown from position 68 to 1417 in SEQ ID NO: 3; and d) afragment of (a), (b) or (c) that has alpha-amylase activity.
 2. Thepolynucleotide of claim 1, wherein the polypeptide is an artificialvariant comprising an amino acid sequence that has one or moretruncation(s), and/or at least one substitution, deletion, and/orinsertion of an amino acid as compared to amino acids 22 to 450 of SEQID NO:
 4. 3. The polynucleotide of claim 1, wherein the polypeptidecomprises an amino acid sequence which has at least 70% identity withamino acids 22 to 450 of SEQ ID NO:
 4. 4. The polynucleotide of claim 1,wherein the polypeptide comprises the amino acids 22 to 450 of SEQ IDNO:
 4. 5. The polynucleotide of claim 1, wherein the polypeptideconsists of the amino acids 22 to 450 of SEQ ID NO:
 4. 6. Thepolynucleotide of claim 1, wherein the polypeptide comprises an aminoacid sequence which has at least 70% identity with the polypeptideencoded by the amylase encoding part of the nucleotide sequence insertedinto a plasmid present in the E. coli host deposited under the BudapestTreaty with DSMZ under accession number DSM
 15334. 7. The polynucleotideof claim 6, wherein the polypeptide comprises the amino acid sequenceencoded by the amylase encoding part of the nucleotide sequence insertedinto a plasmid present in the E. coli host deposited under the BudapestTreaty with DSMZ under accession number DSM
 15334. 8. The polynucleotideof claim 6, wherein the polypeptide consists of the amino acid sequenceencoded by the amylase encoding part of the nucleotide sequence insertedinto a plasmid present in the E. coli host deposited under the BudapestTreaty with DSMZ under accession number DSM
 15334. 9. The polynucleotideof claim 6, wherein the polypeptide is an artificial variant whichcomprises an amino acid sequence that has one or more truncation(s),and/or at least one substitution, deletion, and/or insertion of an aminoacid as compared to the amino acid sequence encoded by the amylaseencoding part of the nucleotide sequence inserted into a plasmid presentin the E. coli host deposited under the Budapest Treaty with DSMZ underaccession number DSM
 15334. 10. A nucleic acid construct comprising apolynucleotide of claim 1 operably linked to one or more controlsequences that direct the production of the polypeptide in a suitablehost cell.
 11. A recombinant expression vector comprising a nucleic acidconstruct of claim
 10. 12. A recombinant host cell comprising a nucleicacid construct of claim
 10. 13. A method for producing a polypeptidehaving alpha-amylase activity, the method comprising: (a) cultivating arecombinant host cell of claim 12 under conditions conducive forproduction of the polypeptide; and (b) recovering the polypeptide.
 14. Amethod of producing an enzymatically modified starch derivative, whereina polypeptide having alpha-amylase activity produced according to amethod of claim 13 is used for liquefying and/or saccharifying starch.15. A method of producing high maltose syrups, wherein a polypeptidehaving alpha-amylase activity produced according to a method of claim 13is used for liquefying starch.
 16. A process for producing an alcoholcomprising liquefying a starch with a polypeptide having alpha-amylaseactivity produced according to a method of claim
 13. 17. An isolatedpolypeptide having alpha-amylase activity, selected from the groupconsisting of: a) a polypeptide with at least 70% sequence identity withamino acids 22 to 450 of SEQ ID NO: 4; b) a polypeptide encoded by apolynucleotide with at least 70% sequence identity with the sequence ofnucleotides 68 to 1417 of SEQ ID NO: 3; and c) a fragment of (a) thathas alpha-amylase activity.
 18. The polypeptide of claim 17, comprisingamino acids 22 to 450 of SEQ ID NO:
 4. 19. The polypeptide of claim 17,consisting of the amino acids 22 to 450 of SEQ ID NO: 4.